Recent outbreaks of “sleeping disease” have caused mass mortality in cultured mud crab and widespread economic loss in southeast China. Two novel pathogens (Mud Crab Reovirus, MCRV and Mud Crab Dicistrovirus, MCDV-1) have been isolated from crab exhibiting “sleeping symptoms”. Thus, there is an urgent need for the accurate and early detection of this pathogen to facilitate disease control. We developed a duplex nested-PCR protocol for the detection of MCRV and MCDV-1 in . Two pairs of primers targeting the conserved regions of the two viruses genes were designed using were ReoF (5′-GCAAATTGAACTACTACTACTTA-3′) and ReoR (5′-GATTCCTATTGTCAACTATCTCA-3′), and the inner primers were ReoNF (5′-ACTCATAGAGCAGTCATGGG-3′) and ReoNR (5′-ATATCGTCAGAATGTC GTTC-3′). The outer primers for MCDV-1 were DicF (5′-GCACTGGGTACTCTTCCTG-3′) and DicR (5′-AC ACCTACCAAAGCCCTAC-3′), and the inner primers were DicNF (5′5′-GGATACTATGGATGATGTTTC-3′) and DicNR (5′- ACAAAATACCAGATAAAGCAA-3′). The PCR products were differentiated by size. The first PCR was carried out using a PCR mix consisting of 1 μL DNA, 1 μL MCRV outer primer (ReoF/ReoR, 0.5 µmol/L), 0.4 μLO (to a final volume of 20 μL). The thermal cycle was: 94 for 10 min. The nested PCR was carried out using the same mix with 0.2 μL template DNA (the product of the first PCR), 0.8 μL MCRV inner primer (ReoNF/ReoNR, 0.4 µmol/L), and 0.8 μL MCDV-1 inner primer (DicNF/DicNR, 0.4 µmol/L). The conditions were the same as in the first PCR, except theannealing temperature was 53. We evaluated the specificity and sensitivity of the duplex nested-PCR. The assay did not exhibit any cross-reactivity with abalone shriveling syndrome-associated virus (AbSV), acute virus necrobiotic virus (AVNV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), fish nervous necrosis virus (NNV), turbot viral reddish body iridovirus (TRBIV), or white spot syndrome virus (WSSV). We used a standard recombinant plasmid for each virus in the and MCRV/MCDV-1 served as the position control. The detection limit was 10¹ copies of the viral genome for the two viruses. Our results suggest that this technique is a rapid, reliable, and cost effective tool for identification of crab virus with high sensitivity, high specificity. The assay could also be applied as a rapid diagnostic tool in clinical samples in which MCRV or MCDV-1 infection is suspected and differential diagnosis is required.